L .E. Moore et al. J. Exp. Mar. Biol. Ecol. 244 2000 131 –143
133
2. Materials and methods
Five experiments were carried out on three species of penaeid prawn in size classes from postlarva to adult Table 1. The first two experiments were preliminary studies
Table 1 to obtain an indication of the magnitude of the response to several days of starvation. Then two more rigorously controlled experiments were carried out to measure
the effects of starvation in juvenile prawns Table 1. In the final experiment, feed was rationed relative to the intake of the control group, which was fed to satiation, to assess
the sensitivity of both the response of the animals and the measuring techniques that were used Table 1.
2.1. Preliminary experiments — postlarva P. monodon and adult P. semisulcatus In Experiment 1, about 100 postlarval P. monodon between 4 and 10 mg wet weight
2–3 mm carapace length, CL were obtained from a prawn hatchery in mid-summer. They were kept in a 390 3 580 3 300 mm plastic mesh basket lined with 2-mm mesh
and suspended in a 7000-l seawater tank. The water temperature was maintained at
Table 1 Summary details of the prawns Penaeus spp. used in experiments to investigate potential indices of
a
nutritional condition: CL and wet weight were measured at the end of the experiment Prawn size
Wet weight CL
Treatments Duration of
NC and species
mm experiment days
Parameters Experiment 1
Postlarval 4–10 mg
2–3 Starved
5 WBL
P. monodon Experiment 2
Adult 28–43 g
32–38 Starved
4 BRI
P. semisulcatus Blood lipid
DGL Experiment 3
Large juvenile 0.3–1.2 g
7–12 Starved
6 WBL
P. semisulcatus or fed
BRI Experiment 4
Small juvenile 20–120 mg
3–6 Starved
3.5 WBL
P. esculentus or fed
Experiment 5 Large juvenile
0.5–1.3 g 10–14
Starved or 6
WBL P. monodon
fed at 25, BRI
50, 75 and 100 satiation
a
NC, nutritional condition; CL, carapace length; WBL, whole body lipid; BRI, blood refractive index; DGL, digestive gland lipid.
134 L
.E. Moore et al. J. Exp. Mar. Biol. Ecol. 244 2000 131 –143
28618C. The prawns were starved for 5 days. About 15 prawns were taken from the basket and frozen on their arrival at the laboratory 0 day sample. Similar samples of
seven prawns were taken at 24-h intervals for 5 days for analysis of WBL content. In Experiment 2, 16 adult male P. semisulcatus 28–43 g; 32–38 mm CL trawled
from the Gulf of Carpentaria, Australia 128409S, 1418409E were placed in a 10 000-l tank at the Cleveland laboratory. They were fed to excess on a diet of fresh, frozen
invertebrates squid, Loligo sp.; mussel, Perna canaliculus; and prawn, Metapenaeus bennettae for 8 weeks, until the start day 0 of the starvation experiment. On each of
days 0, 2 and 4 after the cessation of feeding, a sample of five or six prawns in intermoult stage was taken. The moult stage, wet weight and CL of each prawn were
recorded. The BRI, blood lipid content and digestive gland lipid content of these prawns were determined.
2.2. Experiment 3 — juvenile P. semisulcatus About 100 juvenile P. semisulcatus of 6–8 mm CL were trawled from the Embley
River, Gulf of Carpentaria, Australia 128429S, 1418539E in early summer and flown to the Cleveland laboratory. They were placed in two 300-l tanks and acclimatised to
laboratory conditions for 12 days. During this time, they were fed to excess a commercial prawn feed formulated for P. japonicus; small pieces of fresh, frozen prawn
muscle M. bennettae, and a natural food supplement see below.
The natural food supplement comprised strips of shade-cloth, 500 3 200 mm deep, that had been suspended in an outdoor, 300-l tank. The tanks, which had been inoculated
with about 9 l of mud and seagrass obtained from a nearby seagrass bed, contained large numbers of microbenthic organisms, including amphipods and copepods. The tank was
provided with a constant flow of seawater 23–278C and fertilised weekly with a
slow-release fertiliser 125 g of Osmocote , Scotts-Sierra Horticultural Products to maintain an active culture of micro- and macro-algae. After 3 weeks the shade-cloth was
covered with epiphitic algae and had been colonised by a variety of small invertebrates. Strips of shade-cloth, 50 3 200 mm deep, were cut from the sheets in the outdoor tank
and suspended in each prawn tank or cage to provide a natural food supplement. These were replaced every second day as the biota was grazed down.
After the acclimatisation period, the prawns were placed individually in cylindrical cages 250 mm high 3 150 mm diameter of 6-mm polyethylene mesh with a solid PVC
cap and a removable 390-mm mesh base underneath the polyethylene mesh base. Eleven cages were placed in each of six 300-l tanks. Throughout the experiment, the flow rate
of seawater through the tanks was kept at 1.7 l min, and the water temperature was maintained at 28618C.
Initially all the caged prawns were fed to excess daily. After 5 days, the prawns in three tanks continued to be fed to excess, while those in the remaining three tanks were
starved. On day 0, seven fed prawns were randomly sampled, and on day 1 seven starved animals were sampled. Thereafter, on days 2, 4 and 6, seven fed and seven
starved prawns were randomly sampled. Each prawn was weighed, its CL and BRI measured and then it was immediately placed in a glass vial and frozen at 2 508C for
WBL determination.
L .E. Moore et al. J. Exp. Mar. Biol. Ecol. 244 2000 131 –143
135
2.3. Experiment 4 — Juvenile P. esculentus About 150 small juvenile P. esculentus of 2–3 mm CL were trawled from the Embley
River, and flown to the Cleveland laboratory. Eighty prawns were placed individually in 400-ml beakers full of seawater and covered with cotton netting. The prawns were kept
in a temperature-controlled room to maintain the water in the beakers at 28618C.
Prawns were fed to excess on a mixture of Fripak 300 1 ultra postlarval feed, fresh
diatoms Chaetoceros muelleri and live brine shrimp Artemia salina. The water in the beakers was changed daily.
After 3 weeks, the prawns had grown to between 3 and 6 mm CL. Five randomly selected prawns were taken for the day 0 sample. Half of the remaining prawns
continued to be fed as before and the others were starved. At 12-h intervals thereafter until 84 h, five fed and five starved prawns were sampled: individuals were weighed and
measured, then placed in glass vials and frozen at 2 508C for WBL determination. The BRI was not measured because sufficient blood cannot be drawn from these prawns to
give a reliable reading on the serum refractometer Atago Optical Works.
2.4. Experiment 5 — juvenile P. monodon About 7000 13-day-old postlarval P. monodon were flown from Great Barrier Reef
Marine Hatchery, Bingil Bay, Australia to the Cleveland laboratory in late summer. They were fed initially on diatoms C. muelleri
, live brine shrimp A. salina and Fripak 300 1 ultra postlarval feed. As they grew larger their diet was gradually changed over to
commercial prawn feeds, initially a crumble 1.0 mm diameter formulated for P. japonicus
, and then a starter diet 1.5 mm diameter formulated for P. monodon. After 4 weeks at the laboratory, 80 prawns 0.39–0.86 g were placed individually in the cages
described in Section 2.2. Sixteen cages were placed in each of six 300-l tanks. Seawater flowed through the tanks at 1.7 l min, and the water temperature was maintained at
28618C throughout the experiment.
The caged prawns were fed to satiation once daily on P. monodon starter diet. For the first 8 days, each individual’s feed intake was closely monitored and the feed ration
adjusted to determine the satiation amount. After 8 days in the cages, seven prawns were selected randomly for the initial sample day 0. On day 1, groups of prawns were either
fed to satiation, starved 0 treatment or fed proportions of the satiation ration 25, 50 and 75. On days 3 and 6, seven or eight prawns were randomly selected from each
treatment and their wet weight, CL and BRI were measured, and the WBL in each prawn was determined.
2.5. Measurements and analyses All prawns were weighed individually after being blotted dry on tissue paper.
Postlarvae were weighed on a microbalance to 0.01 mg; juveniles on an analytical balance to 0.1 mg and adults on a top-loading balance to 0.05 g. The CL of the
postlarvae was measured using a microscope fitted with a calibrated ocular micrometer, while the CL of juveniles and adults was measured with dial callipers.
136 L
.E. Moore et al. J. Exp. Mar. Biol. Ecol. 244 2000 131 –143
A sample of blood, between 10 and 700 ml, depending on the size of the prawn, was taken from the pericardial sinus with a disposable 1-ml syringe fitted with a 0.4 3 13
mm needle. The BRI was measured immediately after the sample was taken, using a clinical serum refractometer Atago Optical Works. A 3 sodium chloride solution was
used as a reference, with its refraction edge adjusted to the 1.334 graduation on the refractive index scale. Where the blood lipid was to be determined, the remainder of the
blood sample was frozen at 2 508C until analysed.
Lipids were extracted from individual whole postlarva and juvenile prawns and from the blood and digestive gland of adult prawns in chloroform–methanol 2:1, v v Folch
et al., 1957. The weight of lipid in the extract was determined gravimetrically to the nearest 0.01 mg on a microbalance. The WBL or digestive gland lipid content of each
prawn were expressed as milligrams of lipid per gram wet weight of prawn mg g, while the lipid content of blood in adult prawns was expressed as milligrams of lipid per
millilitre of blood mg ml.
2.6. Data analysis For experiments 1 and 2, the means and standard errors of each parameter were
calculated for each period of starvation. A one-way analysis of variance was used to detect significant differences between the treatments period of starvation for each of
the parameters, and contrasts were made between all possible pairs of treatments. For experiments 3, 4 and 5, the means and standard errors of each parameter BRI and or
WBL for each treatment feeding level were plotted against time, and the relationship between the parameter and time was examined by using linear or quadratic models for
each treatment. The slopes of the lines were tested to see whether they differed significantly from zero. Paired t-tests were also used to test for differences in the
parameters between treatments at each sampling time of the experiment. Correlations between BRI and WBL of fed and starved prawns and of prawns fed between satiation
and starvation Experiment 5 were determined.
3. Results
